1. Field of the Invention
This invention is directed to short peptides that, when associated with MHC molecules for presentation to T cells, produces a stable, non-zero background, immune response from the T cells. The invention is also directed to complexes comprising the peptides and MHC molecules, and complexes coupled with chromatography and other beads. Further, the invention is directed to methods of quantitating immunological assays with peptides and beads of the invention, and methods of identifying new peptides that produce a stable, non-zero background immune response.
2. Description of the Background
One of the critical aspects for the successful evaluation of clinical trials for the immunotherapy of cancer is the ability to monitor critical parameters of the ongoing in vivo immune responses elicited by the vaccine. The classical assays for the immunological monitoring of individuals receiving peptide vaccines have consisted predominantly of in vitro cell culture-based assays such as proliferation, cytokine secretion into supernatants (ELISA), cytotoxicity and antibody responses. The in vitro manipulation of the patient's peripheral blood cells in these assays and the ‘real-time distance’ from acquisition of the blood sample prior to gaining any data have made it difficult to appreciate valuable and directly relevant insights about the vaccine and the resulting immune response. Newly developed technology along with improved reagents and protocols are now available for a more rapid and less manipulated assessment of immunological responses.
MHC molecules are classified as either Class I or Class II molecules. Class II MHC molecules are expressed primarily on cells such as B lymphocytes, macrophages, etc. Class II MHC molecules are recognized by CD4-helper T lymphocytes and induce proliferation (growth or reproduction of similar cells) of helper T lymphocytes and amplification of the immune response to the particular immunogenic peptide that is displayed. Class I MHC molecules are expressed on almost all nucleated cells and are recognized by CD8 cytotoxic T lymphocytes (CTLs), which then induce lysis of the ‘foreign’ antigen-bearing cells. CTLs are particularly important in tumor rejection and in fighting viral infections. The CTL recognizes the antigen in the form of a peptide fragment bound to the MHC Class I molecule, rather than the intact foreign antigen itself. The antigen is normally derived from endogenously synthesized proteins within the cell that are then degraded into small peptide fragments in the cytoplasm.
Immunogenic peptides having binding motifs for MHC Class I molecules are typically between about 8 and about 11 amino acid residues and comprise conserved residues involved in binding proteins encoded by the appropriate MHC allele. Epitopes on a number of potential target proteins can be identified using conserved ‘anchor’ amino acids. Examples of antigens with well defined anchor amino acid residues include peptides from prostate specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigens, malignant melanoma antigen (MAGE-1), Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1), and papilloma virus antigens. The peptides are known to be useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications (see U.S. Pat. No. 6,037,135).
The most established and acclaimed of the new reagents for the detection and measurement of immunological responses are the MHC dimer and tetramer molecules that allow for the specific detection and quantification of antigen-specific T lymphocytes. From an immune response point of view, the enumeration of antigen-specific T cells by dimer and tetramer analysis by itself is unable to provide any information on the functional immunological activity associated with these cells. However, when used in combination with improved intracellular staining protocols and the rapid stimulation of peripheral blood cells with minimal manipulation, dimer and tetramer analysis has the potential to allow for the detection of cytokine secretion within these cells.
There are generally two methods for the detection of cytokine secretion by antigen-specific T cells. The first is the intracellular cytokine (ICC) secretion by flow cytometry that has the potential to provide both a phenotypic and functional characterization of the cytokine response. A limitation of this method is that it is dependent on the period and conditions of stimulation, and has a rather ‘restrictive’ narrow window of time that is assumed and allowed for the optimal detection of the peak cytokine secretion activity during the staining process.
The second method is where cells are stimulated in culture and the cytokine secretion monitored over a longer period of time by the ELISPOT method, allowing for an extended period of secretion and capture of the secreted cytokine, thereby resulting in enhanced detection of functional activity. Advances in optical instrumentation and digital image analysis software offer promise that a certain level of standardization can be achieved between laboratories using the ELISPOT assay for enumerating cytokine secreting cells in response to specific antigenic peptide stimulation.
Based upon their findings at a recent workshop, the Society of Biological Therapy (SBT) has recommended that future clinical trials should strive to utilize direct methods for the phenotypic (such as tetramer or dimer analysis) and functional analysis of immune cells (such as ICC and ELISPOT) in the immunological monitoring of peptide vaccine clinical trials.
The SBT downgraded the other assays such as the proliferation assay and the cytotoxicity assay largely because of the prolonged in vitro manipulations and culture conditions. However, these assays can also have the potential of being important and informative if the number of in vitro manipulations can be kept to a minimum and stimulatory reagents of manageable consistency can be made available.
It is desirable, therefore, to provide an improved method for detecting and accurately measuring functional activities associated with immune cells during an immune response including use of the proliferation assay and the cytotoxicity assay for immunological monitoring of peptide vaccines.
There are various methods available to isolate or separate biological molecules such as cells, antibodies, antigens, proteins, carbohydrates, nucleic acids, and the like. Magnetic separation techniques typically involve the application of a magnetic field to separate ferromagnetic particles contained within a fluid medium. Magnetic separation of one or more targeted molecules present in a solution comprising a mixed population of molecules is well known and the necessary materials are all commercially available (see U.S. Pat. Nos. 6,110,380 and 6,126,835). More particularly, the separation of target biological molecules was conducted using magnetic particles and a magnetic separation device. However, that magnetic separation was limited to use of a fluid containing a mixed population of biological molecules and magnetic particles coated with a ligand (magnetic separation reagent) having sufficient binding specificity and affinity for the target biological molecule.